Emanuela Grassilli

Multiple Signaling Pathways of the Insulin-Like Growth Factor 1 Receptor in Protection from Apoptosis

ABSTRACT The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.

TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis.

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double‐stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA‐binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity‐purified protein was identical to that of the RNA‐binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCβII‐dependent FUS phosphorylation. Moreover, suppression of PKCβII activity in BCR/ABL‐expressing cells by treatment with the PKCβII inhibitor CGP53353, or by expression of a dominant‐negative PKCβII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte‐colony stimulating factor receptor (G‐CSFR) and downregulation of interleukin‐3 receptor (IL‐3R) β‐chain expression, and accelerated G‐CSF‐stimulated differentiation. Downregulation of FUS expression in BCR/ABL‐expressing 32Dcl3 cells was associated with suppression of growth factor‐independent colony formation, restoration of G‐CSF‐induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.

Inhibition of apoptosis by zinc: A reappraisal

Apoptosis--or programmed cell death--is an active type of cell death, occurring in several pathophysiological conditions. One of the most important characteristics of apoptosis is that cell death is preceded by DNA fragmentation, consequent to the activation of nuclear calcium- and magnesium-dependent endonuclease(s). DNA fragmentation can be inhibited by zinc ions. By using several techniques, such as DNA agarose gel electrophoresis, cytofluorimetric analysis of DNA content and of cell cycle, 3H-thymidine incorporation and trypan blue dye exclusion test, we show that zinc, despite completely inhibiting DNA fragmentation and the consequent loss of nuclear DNA content, does not protect rat thymocytes from spontaneous or dexamethasone-induced death. Our data also suggest that DNA fragmentation, although characteristic, is not a critical event for thymocyte death of apoptotic type.

In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death.

Two hours after a single intraperitoneal injection of dexamethasone (20 micrograms/Kg b.w.) into adult male rats, a typical ladder of DNA fragments was detectable upon separation on agarose gels of DNA from thymocytes. This became maximally evident at 4 hours. Accumulation of sulfated glycoprotein-2 (SGP-2) mRNA, whose rate of expression has been associated to the processes of programmed cell death, preceded the appearance of DNA degradation, starting to increase as early as 30 min after steroid injection, and maintained higher than controls until 8 hrs; a different time course was shown by changes in the levels of beta-actin mRNA. In the spleen, under the same conditions, the SGP-2 message also increased at 30 min, prior to DNA fragmentation, but decreased thereafter below the control value.

BCR-ABL prevents c-jun-mediated and proteasome-dependent FUS (TLS) proteolysis through a protein kinase CbetaII-dependent pathway.

ABSTRACT The DNA binding activity of FUS (also known as TLS), a nuclear pro-oncogene involved in multiple translocations, is regulated by BCR-ABL in a protein kinase CβII (PKCβII)-dependent manner. We show here that in normal myeloid progenitor cells FUS, although not visibly ubiquitinated, undergoes proteasome-dependent degradation, whereas in BCR-ABL-expressing cells, degradation is suppressed by PKCβII phosphorylation. Replacement of serine 256 with the phosphomimetic aspartic acid prevents proteasome-dependent proteolysis of FUS, while the serine-256-to-alanine FUS mutant is unstable and susceptible to degradation. Ectopic expression of the phosphomimetic S256D FUS mutant in granulocyte colony-stimulating factor-treated 32Dcl3 cells induces massive apoptosis and inhibits the differentiation of the cells escaping cell death, while the degradation-prone S256A mutant has no effect on either survival or differentiation. FUS proteolysis is induced by c-Jun, is suppressed by BCR-ABL or Jun kinase 1, and does not depend on c-Jun transactivation potential, ubiquitination, or its interaction with Jun kinase 1. In addition, c-Jun-induced FUS proteasome-dependent degradation is enhanced by heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and depends on the formation of a FUS-Jun-hnRNP A1-containing complex and on lack of PKCβII phosphorylation at serine 256 but not on FUS ubiquitination. Thus, novel mechanisms appear to be involved in the degradation of FUS in normal myeloid cells; moreover, the ability of the BCR-ABL oncoprotein to suppress FUS degradation by the induction of posttranslational modifications might contribute to the phenotype of BCR-ABL-expressing hematopoietic cells.

Studies on the relationship between cell proliferation and cell death: Opposite patterns of SGP-2 and ornithine decarboxylase mRNA accumulation in pha-stimulated human lymphocytes

To assess the relationship between cell proliferation and cell death, the mRNA accumulation of ornithine decarboxylase (ODC) and sulfated glycoprotein 2 (SGP-2) were measured in human peripheral blood lymphocytes (HPBL) 2-6 hours after stimulation with phytohemagglutinin (PHA). ODC is the rate limiting enzyme of polyamines biosynthesis and its early induction in mitogen-stimulated lymphocytes has been reported. On the other hand, SGP-2, a glycoprotein present in most mammalian tissues, is induced in classical models of apoptosis, such as dexamethasone-treated thymocytes. Indeed, a consistent amount of SGP-2 mRNA in quiescent HPBL, an early and progressive decrease of SGP-2 mRNA and a parallel increase of ODC mRNA accumulation, were observed, in PHA-stimulated HPBL, suggesting that concomitant repression of SGP-2 and induction of ODC genes contribute for the cell entering the cell cycle.

Cell Proliferation and Cell Death in Immunosenescence

Aging is characterized by a variety of alterations that occur in most organs and cell types. Loss of proliferative vigor is considered a marker of the aging process and is related to the Hayflick phenomenon, that is, the limited number of replications that normal cells can undergo. A correlation between proliferative capability and maximum lifespan in different species, and an inverse correlation between proliferative capability and donor age have been dem~nstrated.l-~ Thus, a decreased capability to proliferate may be considered a characteristic of cellular senescence. In a recent paper we argued that an intriguing relation exists among cellular senescence, tumor growth, and 10ngevity.~ All these phenomena are deeply related to programmed cell death or apopto~is.~ A possible scenario is the following: cells may be equipped with genes that actively promote cellular senescence, thus controlling cell death in order and escaping transformation.68 This situation is balanced by other genes responsible for survival and viability, as depicted in FIGURE 1. Circumstantial evidence suggests that cellular senescence may be considered a peculiar type of cell differentiation whose biological function is to counteract uncontrolled cell proliferation. (For an example, see ref. 9.) From this point of view, cellular senescence can be considered one of the most important mechanisms in avoiding the continuous onset of tumors. The most effective evolutive way for a cell to control neoplastic growth is likely to set up genes that promote apoptotic cell death.1s24

Exposure to 900 MHz Radiofrequency Radiation Induces Caspase 3 Activation in Proliferating Human Lymphocytes

Abstract Palumbo, R., Brescia, F., Capasso, D., Sannino, A., Sarti, M., Capri, M., Grassilli, E. and Scarfì, M. R. Exposure to 900 MHz Radiofrequency Radiation Induces Caspase 3 Activation in Proliferating Human Lymphocytes. Radiat. Res. 170, 327– 334 (2008). In this study, the induction of apoptosis after exposure to 900 MHz radiofrequency radiation (GSM signal) was investigated by assessing caspase 3 activation in exponentially growing Jurkat cells and in quiescent and proliferating human peripheral blood lymphocytes (PBLs). The exposure was carried out at an average specific absorption rate of 1.35 W/kg in a dual wire patch cell exposure system where the temperature of cell cultures was accurately controlled. After 1 h exposure to the radiofrequency field, a slight but statistically significant increase in caspase 3 activity, measured 6 h after exposure, was observed in Jurkat cells (32.4%) and in proliferating human PBLs (22%). In contrast, no effect was detected in quiescent human PBLs. In the same experimental conditions, apoptosis was also evaluated in Jurkat cells by Western blot analysis and in both cell types by flow cytometry. To evaluate late effects due to caspase 3 activity, flow cytometry was also employed to assess apoptosis and viability 24 h after radiofrequency-radiation exposure in both cell types. Neither the former nor the latter was affected. Since in recent years it has been reported that caspases are also involved in processes other than apoptosis, additional cell cycle studies were carried out on proliferating T cells exposed to radiofrequency radiation; however, we found no differences between sham-exposed and exposed cultures. Further studies are warranted to investigate the biological significance of our findings of a dose–response increase in caspase 3 activity after exposure to radiofrequency radiation.

SGP-2, apoptosis, and aging.

In previous papers we argued that the elimination of heavily damaged cells can be considered a cellular defense mechanism that can be particularly important in understanding the aging proccss at the cellular Indeed, it can be speculated that cellular senescence, characterized by terminal differentiation and cell death, plays an important role in counteracting the natural tendency of cells to undergo transformation. This hypothesis can explain why all cells are apparently equipped with a genetic program devoted to programmed cell death.2 The biochemical pathway(s) leading to programmed czll death (apoptosis) and the molecular mechanisms underlying this process are still poorly understood. The activation of a Ca2+, Mg2+-dependent endogenous endonuclease that cuts DNA into discrete multiples of a nucleosomal unit (e.g., 180-bp subunit) seems to play a pivotal role during apoptosis.3 One of the early events following the activation of the cell death program is the increase in intracellular Ca2+. This is very similar to what happens during the early steps of cell proliferation. Induction of c-fos and c-niyc oncogenes seems to be another feature shared by these two opposite phenomena, as reported by Buttyan et aL4 This suggests that specific cellular events may act simultaneously 011 the two processes, leading to opposite biological end points2 To test this hypothesis we focused our attention on the expression of sulfated glycoprotein 2 (SGP-2) mRNA in several systems undergoing cell death, cell proliferation, or aging processes.54

Effects of Pulsed Electromagnetic Fields on the Proliferation of Lymphocytes from Aids Patients, HIV-Seropositive Subjects, and Seronegative Drug Users

The effect of the exposure of mitogen-stimulated lymphocytes from subjects infected by human immunodeficiency virus to extremely low frequency pulsed electromagnetic fields (PEMFs) was studied, by evaluating the incorporation of tritiated thymidine, the expression of IL-2 receptor, and the amount of activated T lymphocytes. Four groups of subjects were considered patients with acquired immunodeficiency syndrome (AIDS), asymptomatic seropositive subjects, seronegative drug users, and young healthy controls. PEMFs increased cell proliferation only in the group of healthy controls, as measured at the 72nd hour of culture, but an increase in the number of activated T lymphocytes was observed by cytofluorimetric analysis after 18 hrs of PEMF exposure in cultures from AIDS patients.